Upper limb muscle stiffness and spasticity after stroke are associated with reduced functional independence and a significant increase in health care costs. If left untreated, it leads to joint contractures, further disability, and increases the economic burden. Spasticity develops as a result of central nervous system injury, which leads to a velocity-dependent increase in stretch reflexes because of decreased cortical influence on the spinal cord. However, secondary changes within the muscles and connective tissue also contribute to muscle stiffness that increase the passive resistance to stretch. The precise non-neural mechanisms and their interaction with neural mechanisms are not well understood, and there are currently no approved medications for treatment of changes in muscle mechanical properties. We proposed the hyaluronan hypothesis, which postulates that the accumulation of hyaluronan within the extracellular matrix (ECM) of muscles promotes the development of muscle stiffness. Hyaluronan is a high molecular weight glycosaminoglycan (GAG) that acts as a lubricant to facilitate intramuscular and intermuscular sliding under physiological conditions. However, when its concentration is increased because of paresis and reduced mobility, it aggregates and makes the ECM hyper-viscous, which can lead to decreased sliding of the muscle fibers and fascicles, muscle shortening, and increased muscle stiffness. Untreated, the increased concentration of hyaluronan can lead to subsequent fibrosis and contracture. We have found that intramuscular injections of the FDA-approved enzyme hyaluronidase, which hydrolyzes long-chained hyaluronan polymers to smaller polymers, can reduce muscle stiffness and increase passive and active range of motion in patients with moderately-severe upper limb spasticity within a few days. Importantly, this effect persists for at least 3 months. These results suggest that reducing muscle hyaluronan concentrations with hyaluronidase is a feasible treatment for muscle stiffness. We now propose to identify the potential mechanisms and test the efficacy of using human recombinant hyaluronidase for treating muscle stiffness. Our central hypothesis is that hyaluronidase will enhance upper limb motor outcomes by modulating peripheral non-neural mechanisms, and reducing the GAG content and viscosity in muscles. We will conduct a proof-of-principle double-blind, randomized, placebo-controlled trial of human recombinant hyaluronidase injections in patients with post-stroke upper limb muscle stiffness to test this hypothesis. The specific aims are to: test the effect of hyaluronidase on upper limb motor outcomes (Aim 1); evaluate the effect of hyaluronidase on neural and non-neural components of muscle stiffness (Aim 2); and elucidate the effect of hyaluronidase on intramuscular GAG content quantified non-invasively by proton T1? relaxation mapping on muscle MRI (Aim 3). At its conclusion, this study will provide mechanistic and behavioral evidence for a novel, practical, and potentially transforming approach to treat muscle stiffness and reduce the burden of disability after neurological injury.